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Experimental Procedures
JMJD2A
Expression
Media:
Starter culture: 2 x LB + 50 µg/mL Kanamycin + 34 µg/mL chloramphenicol
Expression culture: 3 x 1L home-made TB + 50 µg/mL Kanamycin + salts added after autoclaving.
Induction protocol:
Freshly transformed BL21(DE3)-R3-pRARE2 bacteria were used to inoculate starter culture. Each litre of TB in 3L baffled flasks was inoculated with 10 mL of starter culture and grown at 37°C. The protein expression was induced with 0.2 mM IPTG an hour after OD reached OD600 = 0.7 and media cooled to 18°C. The cells were collected by centrifugation and frozen at -80°C.
Extraction Extraction buffers:
Lysis buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 10 mM imidazole, protease inhibitor cocktail set III (Calbiochem), 50U of benzonase (EMD Milipore)
Extraction procedure:
Frozen cell pellets were thawed and resuspended in 200 mL of lysis buffer. The cells were disrupted by high pressure homogenisation (25 kpsi). Cell debris were removed by centrifugation for 60 minutes at 35 000xg
Purification
Column 1
IMAC: HisTrap FF Crude, 1 mL (GE/Amersham Biosciences).
Lysis buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 10 mM imidazole, 5% glycerol.
Wash buffer: 50 mM HEPES, pH 7.5, 500 mM NaCl, 30mM imidazole, 5% glycerol.
Elution buffer 50 mM HEPES pH 7.5, 500 mM NaC, 250 mM imidazole, 5% glycerol.
Procedure:
The cell extract was loaded on the column at 1 mL/minute on an AKTA-express system (GE/Amersham). The column was then washed with 30 column volumes of lysis buffer, 10 volumes of wash buffer, and then eluted with elution buffer. The eluted peak of A280 was automatically collected
Column 2: Ion exchange: 2 x 1ml HiTrap HP Q ion exchange column (GE/Amersham Biosciences).
Low salt buffer: 25 mM HEPES pH 7.5, 50 mM NaCl,
High salt budder: 25 mM HEPES pH 7.5, 1M NaCl, 5% glycerol
Dilution buffer: 25mM HEPES pH 7.5
Procedure:
The eluted fractions from the Ni-affinity Histrap column were pooled and diluted 10x to 50mM NaCl concentration, then loaded on ion exchange columns at flow of 1ml/min. Both peaks were eluted at salt concentration between 150-200mM. Glycerol and NaCl were added during concentration procedure to reach final buffer formula of 25mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol.
Concentration and storage
The protein was concentrated using an Amicon Ultracel centrifugal concentrator (10 kDa MWCO) to 1 mg/ml by A280 and predicted extinction coefficient. Stock was concentrated to 3mg/ml and all samples were frozen in liquid nitrogen followed by storage in -80°C.
Mass spec
The mass determined for JMJD2AA-p084 was 44266 Da, in agreement with the predicted mass for the his-tagged protein. Protein is also modified by N-glucuronylation and phosphogluconoylation which are typical modifications for His-tagged proteins.
Crystallization
JMJD2A was crystallized in sitting drop plates at 4°C by mixing 100nl of 30 mg/mL protein in 20 mM HEPES pH7.5, 5 % (v/v) glycerol, 500 mM NaCl, 0.5 mM TCEP with 50 nL of reservoir solution containing 0.1 M Bis-Tris pH 5.9, 0.15 M ammonium sulphate, 13% (w/v) PEG3350. Plate-like crystals typically grew within a week. The crystals belong to space group P21212 with two molecules in the asymmetric unit and unit cell dimensions of a=101.0Å, b=149.6 Å, c=57.9Å. The typical resolution limit of the crystals is between 1.9 and 2.5 Å.
JMJD2C
Expression
Media:
Starter culture: SOB + 50 µg/mL Kanamycin + 34 µg/mL chloramphenicol
Expression culture: 6 x 1L home-made TB + 50 µg/mL Kanamycin + phosphate salts added after autoclaving.
Induction protocol:
Freshly transformed BL21(DE3)-R3-pRARE2 bacteria were used to inoculate starter culture. Each litre of TB in 3L baffled flasks was inoculated with 10 mL of starter culture and grown at 37°C, 180rpm. The protein expression was induced with 0.2 mM IPTG at OD600 = 2.5 for 18 h at 18°C. The cells were collected by centrifugation and used directly for purification.
Extraction
Extraction buffers:
Lysis buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 10 mM imidazole, protease inhibitor cocktail set III (Calbiochem)
Extraction procedure:
Pellets were thawed and resuspended in 150 mL of lysis buffer +500U benzonase (Novagene). The cells were disrupted by high pressure homogenisation (25 kpsi). Cell debris were removed by centrifugation for 45 minutes at 55 000xg
Purification
Column 1 IMAC
5ml of Ni-Sepharose 6FF in gravity column (GE/Amersham Biosciences).
Lysis buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 10 mM imidazole, 5% glycerol.
Wash buffer: 50 mM HEPES, pH 7.5, 500 mM NaCl, 30mM imidazole, 5% glycerol.
Elution buffer 50 mM HEPES pH 7.5, 500 mM NaC, 250 mM imidazole, 5% glycerol.
Procedure:
The cell extract was applied to gravity column containing equilibrated resin. Resin washed with 20CV of lysis buffer, 20 CV of wash buffer, and then proteins eluted with elution buffer.
Column 2: Gel filtration and TEV cleavage
Gel Filtration: HiLoad 16/60 Superdex 75prep grade, 120 mL (GE/ Amersham Biosciences).
Buffer: 20mM HEPES, 0.5M NaCl, 5% glycerol, pH 7.5
TEV protease purified in house
Procedure:
Pooled fractions were filtered and loaded on two gel filtration columns (flow 1.2ml/min). Fractions corresponding to monomeric protein were pooled together. 2mg of TEV protease was added per each 50mg of target protein and incubated over-night.
Column 3: Rebinding
Protein rebound to 1ml of Ni-sepharose 6 FF equilibrated in gel filtration buffer. Protein solution was passed twice through gravity column containing resin. Resin washed with 5CV of gel filtration buffer and contaminants eluted with 5CV of elution buffer. F-T fraction and gel filtration fractions contained pure protein and were pooled together.
Concentration and storage
The protein was concentrated using an Amicon Ultracel centrifugal concentrator
(10 kDa MWCO) to 5 mg/ml by A280 and predicted extinction coefficient. Frozen in 200ul aliquots in liquid nitrogen and stored at -80C.
Protein yield: 3.7mg/1L
Quality control
Mass spectrometry detects proper molecular weight and protein is enzymatically active confirmed by FDH assay. Protein remains active for at least two freeze-thaw cycles confirmed by demethylase assay with RapidFire detector.
JMJD2D
Expression
Media:
Starter culture: TB (home made) + 50 µg/mL Kanamycin + 34 µg/mL chloramphenicol
Expression culture: 6 x 1L home-made TB + 50 µg/mL Kanamycin + salts added after autoclaving.
Induction protocol:
Freshly transformed BL21(DE3)-R3-pRARE2 bacteria were used to inoculate starter culture. Each litre of TB in 6L baffled flasks was inoculated with 10 mL of starter culture and grown at 37°C. The incubator with cultures was cooled down to 18°C after OD reached 0.7. Protein expression was induced with 0.1 mM IPTG after one hour. The cells were collected by centrifugation and frozen at -80°C.
Extraction
Extraction buffers:
Lysis buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 10 mM imidazole, protease inhibitor cocktail set III (Calbiochem)
Extraction procedure:
Frozen cell pellets were thawed and resuspended in 200 mL of lysis buffer. The cells were disrupted by high pressure homogenisation (25 kpsi). Cell debris were removed by centrifugation for 60 minutes at 35 000xg
Purification
Column 1: IMAC
Ni-Sepharose 6 FF, 2 mL resin (GE/Amersham Biosciences).
Lysis buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 10 mM imidazole, 5% glycerol.
Wash buffer: 50 mM HEPES, pH 7.5, 500 mM NaCl, 30mM imidazole, 5% glycerol.
Elution buffer 50 mM HEPES pH 7.5, 500 mM NaC, 250 mM imidazole, 5% glycerol.
Procedure:
The cell extract was purified through gravity column containing nickel resin. The column was then washed with 25 column volumes (CV) of lysis buffer, 10 CV of wash buffer, and then eluted with elution buffer.
Column 2: TEV cleavage and Gel filtration
HiPrep Superdex 200 16/60 (GE/Amersham Biosciences).
Buffer : 10 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol.
TEV protease produced in house.
Procedure:
The eluted fractions were pooled and TEV protease was added over-night. The following day protein has been concentrated to 5ml and loaded onto gel filtration column. Fractions corresponding to monomeric peak were pooled together.
Column 3: Nickle rebinding
Protein contaminants were rebound to 250ul of Ni-Sepharose using gravity method. After target protein was collected in flow-through the resin was washed with 10CV of gel filtration buffer, lysis buffer and elution buffer each.
Flow-though and gel filtration fractions were containing target protein.
Concentration and storage
The protein was concentrated using an Amicon Ultracel centrifugal concentrator (30 kDa MWCO) to 7 mg/ml by A280 and predicted extinction coefficient. Frozen in Liquid nitrogen and stored at -80°C.
The purification procedure remains to be optimised.
Crystallization
JMJD2D was crystallized in sitting drop plates at 20°C by mixing 100 nL of 11mg/ml protein in 20 mM HEPES pH7.5, 5 % (v/v) glycerol, 500 mM NaCl, 0.5 mM TCEP with 50 nL of reservoir solution containing 0.1 M
Bis-Tris pH 5.9, 0.15 M ammonium sulfate , 11% (w/v) PEG3350. Bi-pyramidal shaped crystals grew within several days. They belong to space group P43212 with one molecule in the asymmetric unit and typical unit cell dimensions of a=b=72 Å, c=151Å. The typical resolution limit of the crystals is between 1.1 and 1.5 Å.
Assay Conditions
The JMJD2C demethylase assay uses the peptide: biotin-ARTKQTARK(Me3)STGGKAPRKQLA-GGK-Biotin (Histone H3 Lys 9 tri-methyl) as a substrate and relies on detection of H3K9Me2-biotin bound to streptavidin donor beads by a monoclonal anti-H3K9Me2 antibody coupled to protein-A acceptor beads.
Screens use low nM concentrations of JMJD2 (1.0 nM enzyme routinely used in the assay) and nM concentrations of peptide substrate (H3K9Me3 routinely used at 30 nM in assay screens). Assays are performed in 384-well proxiplates and compounds dispensed using ECHO Acoustic dispensing.
Experimental Conditions
Material
Reagent |
Supplier |
Code |
HEPES Free Acid >99.5% |
Gibco Life Technologies |
11344-041 |
H3(1-21)K9Me3 Peptide |
Anaspec |
64360 |
(NH4)2[Fe(SO4)2].6H20 |
Sigma-Aldrich |
12304 |
L-Ascorbic Acid |
Sigma-Aldrich |
A5960 |
2-OG (α-ketoglutaric acid) |
Sigma-Aldrich |
K3752 |
Tween-20 |
Fisher Scientific |
BPE-337-500 |
BSA Cohn Fraction V |
Sigma-Aldrich |
**A7030 |
Anti-H3K9Me2 |
Abcam |
Ab1220 |
EDTA |
Fisher Scientific |
BP120 |
NaCl |
VWR Analar Normapur |
27810.364 |
AlphaScreen General IgG |
Perkin Elmer |
6760617c |
384-well proxiplates plus |
Perkin Elmer |
6008280 |
Thermowell Sealing Tape |
Corning |
6570 |
**Critical : Always use this batch of BSA
Alphascreen Beads
Store at 4°C
H3K9Me3 substrate
H3K9Me3-biotin substrate is stored at -80°C in 24 ml aliquots at a concentration of 100 mM.
JMJD2 Enzyme
JMJD2C is stored in 1 mM aliquots at -80°C.
Preparation of Solutions
Assay Buffer:
Prepare fresh every week and filter sterilize through a 0.2 micron filter: 50mM HEPES pH 7.5, 0.01% Tween-20, 0.1% BAS. Store at 4°C
Assay Stop:
Prepare fresh each day. Assay buffer containing 30mM EDTA + 800 mM NaCl (4X final concentration).
Ferrous Ammonium Sulphate (FAS):
Prepare FAS fresh each dat. Make up 400 mM stock solution (156.856 mg/ml)in 20mM HCl and then prepare 2 ml of 1mM FAS in deionized H2O. Store at room temperature.
2-Oxoglutarate (2-OG):
Prepare 2-OG fresh each dat. Make up 10mM stock solution in deionized H2O (1.901 mg/ml) and store at room temperature.
L-Ascorbic Acid (L-AA):
Prepare L-AA fresh each day. Make up 50 mM stock solution in deionized H2O (8.806 mg/ml) and store at room temperature
Substrate Solution
|
|
|
Volume Required For |
||
|
Stock Concentration |
2.0X Concentration |
10 ml of Substrate |
20 ml of Substrate |
30 ml of Substrate |
L-Ascorbic Acid |
50 mM |
200 mM |
40 µl |
80 µl |
120 µl |
FAS |
1 mM |
2.0 mM |
20 µl |
40 µl |
60 µl |
H3K9Me3-Biotin |
100 mM |
0.06 mM |
6.0 µl |
12 µl |
18 µl |
2-OG |
10 mM |
20 mM |
20 µl |
40 µl |
60 µl |
Assay Buffer |
|
|
9914 µl |
14828 µl |
29742 µl |
Protocol
- Calculate the amount of alphascreen beads required for the experiment.
- Prepare the required amount of beads in a dark box at 4X the final concentration:
Alphascreen donor beads and acceptor beads are mixed and pre-incubated with Anti-H3K9Me2 antibody in assay buffer for at least 1 hour before addition to the assay. Make up at 4X final concentration:
Streptavidin Donor 0.08 mg/ml Final in Assay = 0.02 mg/ml
Protein A Acceptor 0.08 mg/ml Final in Assay = 0.02 mg/ml
Anti-H3K9Me2 0.2 mg/ml Final in Assay = 0.05 mg/ml
- Retrieve an aliquot of JMJD2C and 100 mM peptide from the -80°C and place on ice.
- Weigh out Ferrous Ammonium Sulphate (100 -150 mg), 2-Oxoglutarate (2 - 4 mg) and L-Ascorbic Acid (8 -16 mg) in 2.0 ml eppendorf tubes and prepare the solutions as described in section 4.
- Compounds should be supplied in Labcyte 384-LDV (5.0 – 10 ml per well) or Labcyte 384-PP (25 – 45 ml per well) plates at 10 mM concentration in DMSO. IC50 plates are set up using ECHO dose response software and single shot screening plates are set up using ECHO plate reformat software. Transfer compound to proxiplate using ECHO 550 (maximum DMSO in assay should be 1%). A typical 11-point IC50 plate layout for 16 compounds is shown below with column 12 as a DMSO control and column 24 (100 mM 2, 4-PDCA reference compound)
- Dilute JMJD2C enzyme to 2.0 nM in 50 mM HEPES pH 7.5, 0.01% Tween-20, 0.1% BSA and dispense 5.0 ml into columns 1 -24 of a 384-well proxiplate. Dispenses are performed using a Thermo multidrop.
- Incubate the plate for 15 minutes on the bench at room temperature
- Prepare enough 2.0X substrate solution containing L-Ascorbic Acid (200 mM), FAS (2.0 mM), Peptide (60 nM) and 2-OG (20 mM) and dispense 5 ml of substrate into columns 1 -24 of the assay plate. Seal the plate.
- Allow the enzyme reaction to proceed at room temperature for 20 minutes.
- Dispense across the plate 5 ml of assay stop from section 4.2.
- In a dark box, dispense 5 ml of the alphascreen beads prepared in STEP 2 into every well of the assay plate. Seal the plate with an aluminium plate foil.
- Incubate the plate for 120 minutes at room temperature.
- Read the plate on the BMG Labtech Pherastar FS.
- Black, J. C., Atabakhsh, E., Kim, J., Biette, K. M., Van Rechem, C., Ladd, B., Burrowes, P. D., Donado, C., Mattoo, H., Kleinstiver, B. P., Song, B., Andriani, G., Joung, J. K., Iliopoulos, O., Montagna, C., Pillai, S., Getz, G., and Whetstine, J. R. (2015) Hypoxia drives transient site-specific copy gain and drug-resistant gene expression. Genes & development 29, 1018-1031
- Black, J. C., Zhang, H., Kim, J., Getz, G., and Whetstine, J. R. (2016) Regulation of Transient Site-specific Copy Gain by MicroRNA. The Journal of biological chemistry 291, 4862-4871
- Van Rechem, C., Black, J. C., Greninger, P., Zhao, Y., Donado, C., Burrowes, P. D., Ladd, B., Christiani, D. C., Benes, C. H., and Whetstine, J. R. (2015) A coding single-nucleotide polymorphism in lysine demethylase KDM4A associates with increased sensitivity to mTOR inhibitors. Cancer discovery 5, 245-254
- Black, J. C., Manning, A. L., Van Rechem, C., Kim, J., Ladd, B., Cho, J., Pineda, C. M., Murphy, N., Daniels, D. L., Montagna, C., Lewis, P. W., Glass, K., Allis, C. D., Dyson, N. J., Getz, G., and Whetstine, J. R. (2013) KDM4A lysine demethylase induces site-specific copy gain and rereplication of regions amplified in tumors. Cell 154, 541-555
- Shi, L., Sun, L., Li, Q., Liang, J., Yu, W., Yi, X., Yang, X., Li, Y., Han, X., Zhang, Y., Xuan, C., Yao, Z., and Shang, Y. (2011) Histone demethylase JMJD2B coordinates H3K4/H3K9 methylation and promotes hormonally responsive breast carcinogenesis. Proceedings of the National Academy of Sciences of the United States of America 108, 7541-7546
- Kim, T. D., Oh, S., Shin, S., and Janknecht, R. (2012) Regulation of tumor suppressor p53 and HCT116 cell physiology by histone demethylase JMJD2D/KDM4D. PloS one 7, e34618
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Structures of X-ray crystallography fragment hits in complex with KDM4D